Identification and characterization of heteroclitic peptides in TCR-binding positions with improved HLA-binding efficacy

Abstract

The antigenicity as well as the immunogenicity of tumor associated antigens (TAAs) may need to be potentiated in order to break the immunological tolerance. To this aim, heteroclitic peptides were designed introducing specific substitutions in the residue at position 4 (p4) binding to TCR. The effect of such modifications also on the affinity to the major histocompatibility class I (MHC-I) molecule was assessed. The Trp2 antigen, specific for the mouse melanoma B16F10 cells, as well as the HPV-E7 antigen, specific for the TC1 tumor cell lines, were used as models. Affinity of such heteroclitic peptides to HLA was predicted by bioinformatics tools and the most promising ones were validated by structural conformational and HLA binding analyses. Overall, we demonstrated that TAAs modified at the TCR-binding p4 residue are predicted to have higher affinity to MHC-I molecules. Experimental evaluation confirms the stronger binding, suggesting that this strategy may be very effective for designing new vaccines with improved antigenic efficacy.

Fig. 1

Prediction of binding affinity. a, b The predicted binding affinity to the H2-Db molecule of all possible heteroclitic HPV-E7 and Trp2 peptides is shown. The arrow indicates the affinity (nM) of the wt peptide. All the peptides with highest affinity (lower nM values) are on the left of the graph

Fig. 2

Peptide selection. a, b Heteroclitic peptides with affinity higher than the wt, with amino acid substitution in p4, are indicated for both HPV-E7 and Trp2 peptides. The ones selected for the present study are indicated by green arrow. The sequence and predicted binding affinity to H2-Db molecule of each peptide are shown

Fig. 3

Structure of the amino acid residues at p4 in heteroclitic peptides. The structure of the substituting amino acid residues at p4 in the selected heteroclitic peptides is shown, together with the chemical properties of the side chains

Fig. 4

Predicted peptide conformation. a, b The conformation of the wt HPV-E7 and Trp2 peptides, bound to the H2-Db molecule, is shown compared to the LCMV peptide KAVYNFATC (1FG2, protein data bank). The amino acid at the anchor position 5 is highlighted for the three peptides. The presence of the non-polar Phenylalanine (Phe, F) in the Trp2 peptide results in a very poor binding to the H2-Db molecule, compared to the HPV-E7 and LCMV peptides

Fig. 5

Predicted heteroclitic HPV-E7 peptides conformation. a The conformation of the heteroclitic HPV-E7 peptides, bound to the H2-Db molecule, is shown compared to the wt sequence. Contact pattern to the H2-Db are indicated in grey for both front and rear sides. b Prediction of the heteroclitic peptides’ structure presented to the CD8⁺ T cell for interaction with the TCR

Fig. 6

Predicted heteroclitic Trp2 peptide conformation. a The conformation of the heteroclitic Trp2 peptides, bound to the H2-Db molecule, is shown compared to the wt sequence. Contact pattern to the H2-Db are indicated in maroon for both front and rear sides. b Prediction of the heteroclitic peptides’ structure presented to the CD8⁺ T cell for interaction with the TCR

Fig. 7

Binding affinity to the H2-Db molecule. Binding to H2-Db molecule was assessed in TAP-deficient RMA-S murine cells loaded with the indicated peptides. a Mean fluorescence intensity at flow cytometer indicates binding levels of HPV-E7 wt and heteroclitic peptides to the H2-Db molecule at the different concentrations. b Fold-increase (wt = 1) of the binding to the H2-Db molecule of each heteroclitic peptides at the different concentrations. c Mean fluorescence intensity at flow cytometer indicates binding levels of wt and heteroclitic Trp2 peptides to the H2-Db molecule at the different concentrations. d Fold-increase (wt = 1) of the binding to the H2-Db molecule of each heteroclitic peptides at the different concentrations